1. Field
The invention relates to the fields of biology and medicine. Specifically, the invention relates to the prevention, diagnosis, and treatment of neurodegenerative diseases, including, but not limited to, Alzheimer's disease.
2. Related Art
Alzheimer's disease (AD) is a progressive and degenerative dementia (Terry, R. D. et al. (1991) “Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment” Ann. Neurol., vol. 30, no. 4, pp. 572-580; Coyle, J. T. (1987) “Alzheimer's Disease” in Encyclopedia of Neuroscience, Ed. G. Adelman, pp 29-31, Birkhäuser: Boston-Basel-Stuttgart). In its early stages, however, AD manifests primarily as a profound inability to form new memories (Selkoe, D. J. (2002) “Alzheimer's disease is a synaptic failure” Science, vol. 298, pp. 789-791). The basis for this specific impact is not known, but evidence favors involvement of neurotoxins derived from amyloid beta (Aβ). Aβ is an amphipathic peptide whose abundance is increased by mutations and risk factors linked to AD. Fibrils formed from Aβ constitute the cores of amyloid plaques, which are hallmarks of AD brain. Analogous fibrils generated in vitro are lethal to cultured brain neurons. These findings provided the central rationale for the original amyloid cascade hypothesis, a remarkably productive theory in which memory loss was proposed to be the consequence of neuron death caused by fibrillar Aβ.
Despite its strong experimental support and intuitive appeal, the original amyloid cascade hypothesis has proven inconsistent with key observations, including the poor correlation between dementia and amyloid plaque burden (Katzman, R. (1988) “Clinical, pathological, and neurochemical changes in dementia: a subgroup with preserved mental status and numerous neocortical plaques” Ann. Neurol., vol. 23, no. 2, pp. 138-144). Particularly telling are recent studies of experimental AD vaccines done with transgenic hAPP mice (Dodart, J. C. et al. (2002) “Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model” Nat. Neurosci., vol. 5, pp. 452-457; Kotilinek, L. A. et al. (2002) “Reversible memory loss in a mouse transgenic model of Alzheimer's disease” J. Neurosci., vol. 22, pp. 6331-6335). These mice provide good models of early AD, developing age-dependent amyloid plaques and, most importantly, age-dependent memory dysfunction. Two surprising findings were obtained when mice were treated with monoclonal antibodies against Aβ: (1) vaccinated mice showed reversal of memory loss, with recovery evident in 24 hours; (2) cognitive benefits of vaccination accrued despite no change in plaque levels. Such findings are not consistent with a mechanism for memory loss dependent on neuron death caused by amyloid fibrils.
Salient flaws in the original hypothesis have been eliminated by an updated amyloid cascade that incorporates a role for additional neurologically active molecules formed by Aβ self-assembly. These molecules are soluble Aβ oligomers. Oligomers are metastable and form at low concentrations of Aβ1-42 (Lambert, M. P. et al. (1998) “Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins” Proc. Natl. Acad. Sci. USA, vol. 95, pp. 6448-6453). Essentially the missing links in the original cascade, Aβ oligomers rapidly inhibit long-term potentiation (LTP), a classic experimental paradigm for memory and synaptic plasticity. In the updated cascade: (1) memory loss stems from synapse failure, prior to neuron death; and (2) synapse failure is caused by Aβ oligomers, not fibrils (Hardy, J. & Selkoe, D. J. (2002) “The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics” Science, vol. 297, pp. 353-356). Recent reports show soluble oligomers occur in brain tissue and are strikingly elevated in AD (Kayed, R. et al. (2003) “Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis” Science, vol. 300, pp. 486-489; Gong, Y. et al. (2003) “Alzheimer's disease-affected brain: presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memory loss” Proc. Natl. Acad. Sci. USA, vol. 100, pp. 10417-10422) and in hAPP transgenic mice AD models (Kotilinek, L. A. et al. (2002) “Reversible memory loss in a mouse transgenic model of Alzheimer's disease” J. Neurosci., vol. 22, pp. 6331-6335; Chang, L. et al. (2003) “Femtomole immunodetection of synthetic and endogenous amyloid-β oligomers and its application to Alzheimer's Disease drug candidate screening” J. Mol. Neurosci., vol. 20, pp. 305-313).
Amyloid beta immunotherapy for Alzheimer's disease has shown initial success in mouse models of AD and in human patients not susceptible to meningoencephalitis. Disclosed herein are monoclonal antibodies against soluble Aβ oligomers (ADDLs). The antibodies distinguish between AD and control human brain extracts. The antibodies identify endogenous oligomers in AD brain slices and also bind to cultured hippocampal cells. The antibodies neutralize endogenous and “synthetic” ADDLs in solution. So-called “synthetic” ADDLs are produced in vitro by mixing purified amyloid β1-42 under conditions that produce ADDLs, see U.S. Pat. No. 6,218,506. One of the antibodies, 20C2, shows high selectivity for 3-24mers, but minimal detection of monomer Aβ peptides. Recognition of ADDLs by 20C2 is not blocked by short peptides that encompass the linear sequence of Aβ 1-42 or by Aβ 1-40. However, binding is blocked by Aβ 1-28, suggesting an epitope based on conformationally unique structures also attained with Aβ 1-28.
AD is a fatal progressive dementia that has no cure at present. Although the molecular basis of the disease is not established, considerable evidence indicates that it is a proteinopathy involving neurotoxins derived from the 42-amino acid peptide amyloid beta (Aβ). A recent revision of the major “amyloid cascade hypothesis” to explain disease progression states that small soluble Aβ oligomers, as well as the larger Aβ fibrils that constitute the core of plaques, are pathogenic (Hardy, J. & Selkoe, D. J. (2002) “The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics” Science, vol. 297, pp. 353-356).
Recent studies have shown that small soluble Aβ oligomers (also called Aβ-derived diffusible ligands or ADDLs) are present in AD brain, increasing up to 70-fold over control subjects (Gong, Y. et al. (2003) “Alzheimer's disease-affected brain: Presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memory loss” Proc. Natl. Acad. Sci. USA, vol. 100, pp. 10417-10422). The very abundance of ADDLs in AD brain suggests their potential for therapeutic drugs or vaccines. Earlier clinical trials of a vaccine have revealed that persons mounting a vigorous immune response to the vaccine exhibited cognitive benefit (Hock, C. et al. (2003) “Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease” Neuron, vol. 38, pp. 547-554). These findings indicate genuine therapeutic promise, despite the unacceptable frequency of CNS inflammation that caused early termination of part of the trial (Birmingham, K. & Frantz, S. (2002) “Set back to Alzheimer vaccine studies” Nat. Med., vol. 8, pp. 199-200).
An alternative to a live vaccine is the development of therapeutic antibodies that target ADDLs without binding monomers or fibrils (Klein, W. L. (2002) “Aβ toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets” Neurochem. Int., vol. 41, pp. 345-352). Previous work has shown that ADDLs are excellent antigens, generating oligomer-selective polyclonal antibodies in rabbits at the very low antigen concentration of ˜50 ug/ml (Lambert, M. P. et al. (2001) “Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies” J. Neurochem., vol. 79, pp. 595-605). Results from tg-mice models also suggest that antibodies can be successful in reversing memory decline (Dodart, J. C. et al. (2002) “Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease” Nat. Neurosci., vol. 5, pp. 452-457).
Immunization of tg mice models of AD with fibrillar amyloid beta protein (Aβ) results in reduction of Aβ deposits in the brain and prevents the formation of this pathology when administered before its formation (Schenk, D. (2002) Amyloid-beta immunotherapy for Alzheimer's disease: the end of the beginning. Nat. Rev. Neurosci. 3(10):824-8; Schenk, D. et al. (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400(6740):173-7). Learning and memory deficits produced in these mice are also reduced or prevented by similar active vaccination with preparations containing fibrillar Aβ (Janus, C. et al. (2000) A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease. Nature 408(6815):979-82; Morgan, D. et al. (2000) A beta peptide vaccination prevents memory loss in an animal model of Alzheimer's disease. Nature 408(6815):982-5). Based on results from animal models, clinical trials were initiated and showed few adverse reactions in Phase 1. However, Phase 2 trials were halted when 6% of the patients developed meningoencephalitis (Birmingham, K. & Frantz, S. (2002) Set back to Alzheimer vaccine studies. Nat. Med. 8(3):199-200; Hock, C. et al. (2003) Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron 38(4):547-54; Orgogozo, J. M. et al. (2003) Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology 61(1):46-54; Schenk, D. (2002) Amyloid-beta immunotherapy for Alzheimer's disease: the end of the beginning. Nat. Rev. Neurosci. 3(10):824-8; Schenk, D. et al. (2004) Current progress in beta-amyloid immunotherapy. Curr. Opin. Immunol. 16(5):599-606). Reports of the clinical outcome of these trials revealed that after 1 year patients producing antibodies that targeted plaques had a slower rate of cognitive decline than those patients that did not produce antibodies (Hock, C. et al. (2003) Antibodies against beta-amyloid slow cognitive decline in Alzheimer's disease. Neuron 38(4):547-54). Post mortem results on two patients showed absent or sparse plaques in the neocortex, with reactive microglia suggesting an effective immune response (Ferrer, I. et al. (2004) Neuropathology and pathogenesis of encephalitis following amyloid-beta immunization in Alzheimer's disease. Brain Pathol 14(1): 11-20; Nicoll, J. A. et al. (2003) Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat. Med. 9(4):448-52).
Alternative approaches to avoid inflammatory responses through the use of therapeutic antibodies are now under development (Agadjanyan, M. G. et al. (2005) Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J. Immunol. 174(3):1580-6; Gelinas, D. S. et al. (2004) Immunotherapy for Alzheimer's disease. Proc. Natl. Acad. Sci. USA 101(Suppl 2):14657-62; Morgan, D. & Gitter, B. D. (2004) Evidence supporting a role for anti-Abeta antibodies in the treatment of Alzheimer's disease. Neurobiol. Aging 25(5):605-8; Schenk, D. et al. (2004) Current progress in beta-amyloid immunotherapy. Curr. Opin. Immunol. 16(5):599-606). It has been established that injections with Aβ-generated monoclonal antibodies produce cognitive improvement in tg mice models of AD. Using an antibody whose epitope targets the center of the Aβ peptide, it was shown that memory deficits can be reversed in PDAPP mice within 24 hours after treatment (Dodart, J. C. et al. (2002) Immunization reverses memory deficits without reducing brain A beta burden in Alzheimer's disease model. Nature Neuroscience 5(5):452-7). Similarly, in Tg2576 mice, memory loss was reversed using an antibody targeting the N-terminus of Aβ (Kotilinek, L. A. et al. (2002) Reversible memory loss in a mouse transgenic model of Alzheimer's disease. J. Neurosci. 22(15):6331-5).
Passive vaccination previously was shown to clear plaques from PDAPP and other tg mice models (Bacskai, B. J. et al. (2002) Non-Fc-mediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy. J. Neurosci. 22(18):7873-8; Bard, F. et al. (2003) Epitope and isotype specificities of antibodies to beta-amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc. Natl. Acad. Sci. USA 100(4):2023-8; Bard, F. et al. (2000) Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat. Med. 6(8):916-9; McLaurin, J. et al. (2002) Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nature Medicine 8(11):1263-9). However, in the studies showing recovery from memory deficits, Aβ plaque burden was not decreased. A likely explanation for cognitive improvement without change in plaque burden is that these therapeutic antibodies immunoneutralize small, soluble oligomers of Aβ, which have been implicated in AD synapse failure (Lacor, P. N. et al. (2004) Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J. Neurosci. 24(45):10191-200). Aβ oligomers form at low doses of Aβ 1-42, block LTP, and specifically attach to synaptic terminals (Lacor, P. N. et al. (2004) Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J. Neurosci. 24(45):10191-200; Lambert, M. P. et al. (1998) Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc. Natl. Acad. Sci. USA 95(11):6448-53; Wang, H. W. et al. (2002) Soluble oligomers of beta amyloid (1-42) inhibit long-term potentiation, but not long-term depression, in rat dentate gyrus. Brain Res. 924(2):133-40; Wang, Q. et al. (2004) Block of long-term potentiation by naturally secreted and synthetic amyloid beta-peptide in hippocampal slices is mediated via activation of the kinases c-Jun N-terminal kinase, cyclin-dependent kinase 5, and p38 mitogen-activated protein kinase as well as metabotropic glutamate receptor type 5. J. Neurosci. 24(13):3370-8). These oligomers (referred to as ADDLs) are elevated in AD brain and CSF and in tg mouse models (Chang, L. et al. (2003) Femtomole immunodetection of synthetic and endogenous amyloid-beta oligomers and its application to Alzheimer's disease drug candidate screening. J. Mol. Neurosci. 20(3):305-13; Georganopoulou, D. G. et al. (2005) Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer's disease. Proc. Natl. Acad. Sci. USA 102(7):2273-76; Gong, Y. et al. Alzheimer's disease-affected brain: presence of oligomeric A beta ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc. Natl. Acad. Sci. USA 2003 100(18): 10417-22).
Given these considerations, oligomers provide an optimum target for therapeutic antibodies. The present invention addresses the need to obtain monoclonal antibodies selective for oligomers (ADDLs). The approaches disclosed herein use as antigen soluble Aβ oligomers (ADDLs) because of their previously demonstrated utility in providing epitopes dependent on quaternary structure in the generation of polyclonal antibodies. This strategy has generated monoclonal antibodies that distinguish between AD and control brains and that neutralize oligomers in solution, characteristics that will be essential for therapeutically useful antibodies.